Insertion of genes into hematopoietic stem cells has several potential therapeutic applications. Needed is an efficient method for gene insertion and sufficient knowledge about gene regulation to allow design of vectors that insure expression of the transferred gene in the appropriate hematopoietic lineage. We have used the mouse model to systematically explore the variables that determine success in using stem cell as a target for gene insertion. Prior work has shown that hematopoietic growth factors are required during in vitro culture for preservation and amplification of primitive hematopoietic cells. The combination of interleukin-3 (IL-3), interleukin-6 (IL-6) and stem cell factor (SCF) results in a 3-5 fold increase in repopulating activity and a 25 fold increase in the number of spleen colony-forming cells (CFU-S) during 6 days of in vitro culture. Administration of SCF to intact animals results in a 5-7 fold increase in repopulating stem cell activity in the whole animal; enlargement of the spleen is accompanied by a marked increase in stem cell numbers in that organ. A combination of centrifugal elutriation, substraction of cells expressing lineage-specific antigens and positive sorting for the c-kit protein (the receptor for SCF) has allowed preparation of cell fractions highly enriched in repopulating cells. One hundred cells are sufficient to achieve long-term reconstitution of all hematopoietic lineages. These highly enriched fractions can be separated into rhodamine-dull and rhodamine-bright cells, a property that reflects mitochondrial number and expression of the multi-drug resistance gene. In future experiments we will determine whether purified fractions having reconstituting activity but lacking primitive progenitors (CFU-S) can be induced into cell cycle by autologous stroma or stromal cell lines expressing transmembrane SCF. By using highly purified cells under defined culture conditions we hope to increase gene transfer efficiency to 100% of repopulating stem cells.